乳酸菌与益生元组合的筛选并利用IPEC-J2细胞模型验证其对肠道屏障的作用

Screening Combination of Lactobacillus spp. and Prebiotics andEvaluating Their Impact on Intestinal Barrier UtilizingIPEC-J2 Cell Model

为开发改善动物肠道健康的绿色添加剂产品,本研究旨在筛选乳酸菌与益生元组合,并探究其对肠道屏障的影响。首先,根据碳水化合物生化鉴定试剂条的发酵结果确定5种乳酸菌[植物乳杆菌177(LP)、唾液乳杆菌KLW001(LSa)和罗伊氏乳杆菌(LRe)、类干酪乳杆菌(LPa)和鼠李糖乳杆菌(LRh)]对应的候选益生元;其次,根据5种乳酸菌和候选益生元[山梨醇(So)、甘露醇(Ma)、鼠李糖(Rh)、低聚木糖(Xyo)、海藻糖(Tr)、棉子糖(Ra)和普鲁兰多糖(Pu)]的短链脂肪酸(SCFA)发酵能力选择合生元组合,将筛选得到的3种合生元组合(LP+Pu、LSa+Pu、LRe+Ra)通过脂多糖(LPS)构建的“肠漏”细胞模型,以测定单层细胞渗透率、跨上皮细胞电阻(TEER)值的变化、肠道紧密连接蛋白[跨膜蛋白-1(Claudin-1)、闭合蛋白(Occludin)和闭锁小带蛋白-1(ZO-1)]的表达,验证合生元对肠道屏障的作用效果。结果表明:LP+Pu组合能发酵产生更多的SCFA,特别是能增加丁酸含量;“肠漏”细胞模型结果表明,LP+Pu组合上调了Claudin-1和ZO-1的mRNA相对表达量,维持了细胞上皮完整性。综上所述,LP+Pu组合可以作为增加SCFA产量、增加肠道紧密连接蛋白表达的合生元,为今后治疗及预防畜禽肠道屏障相关疾病提供新思路。

In order to develop green additive products to improve the intestinal health of animals,this study aimed to screen the synbiotic combination of Lactobacillus spp.and prebiotics,and investigate its influence on the integrity of the intestinal barrier.Initially,the fermentation results using API 50 CHL kit were used to identify five strains of Lactobacillus spp.[Lactobacillus plantarum 177(LP),Lactobacillus salivarius KLW001(LSa),Lactobacillus reuteri KLR3004(LRe),Lactobacillus paracaeei KCTC3510(LPa),and Lactobacillus rhamnosus KCTC5033(LRh)]corresponding to candidate prebiotics.Then,based on the fermentation ability of short-chain fatty acids(SCFA)by the five strains of Lactobacillus spp.and candidate prebiotics[sorbitol(So),mannitol(Ma),rhamnose(Rh),xylo-oligosaccharide(Xyo),trehalose(Tr),raffinose(Ra),and pullulan(Pu)],synbiotic combinations were selected.Three selected synbiotic combinations(LP+Pu,LSa+Pu,and LRe+Ra)were then applied to lipopolysaccharide(LPS)-induced“leaky gut”cell model.The effect of synbiotics on intestinal barrier function was validated by measuring changes in monolayer cell permeability,trans-epithelial electrical resistance(TEER)values,and the mRNA expression of intestinal tight junction proteins[Claudin-1,Occludin,and zonula occluden-1(ZO-1)].Results showed that the combination of LP+Pu exhibited enhanced fermentation capabilities,resulting in higher production of SCFA,particularly an increase in butyric acid content;in the“leaky gut”cell model,the synbiotic consisting of LP+Pu demonstrated an upregulation of mRNA relative expression for intestinal tight junction proteins,specifically Claudin-1 and ZO-1.Furthermore,this synbiotic combination contributed to the preservation of cell epithelial integrity.The results of this study confirm that the combination of LP+Pu can serve as a synbiotic formulation,effectively enhancing the production of SCFA and increasing the expression of intestinal tight junction proteins.These findings present a novel avenue for the treatment and prevention of intestinal barrier-related diseases in livestock and poultry.